Senescence In Human Melanocytes Research Articles

Premature senescence in human melanocytes after exposure to solar UVR: An exosome and UV-miRNA connection.

Ultraviolet radiation (UVR) can play two roles: induce cellular senescence and convert skin melanocytes into melanoma. To assess whether this conversion might rely on melanocytes having to first acquire a senescent phenotype, we studied the effects of physiological doses of UVR (UVA+UVB) on quiescent melanocytes in vitro. Repeated doses of UVR induced these melanocytes into a senescent-like state. Additionally, these cells secrete exosomes with specific miRNAs that differ in quantity from those of the un-irradiated melanocytes. Many of the exosomal miRNAs that were differentially enriched regulated genes comprising a "senescence core signature" and encoding factors of the senescence-messaging secretome (SASP), while a subset of the differentially reduced miRNAs targeted DNA repair genes that have been experimentally shown to be repressed in senescent melanocytes. Thus, the selection of specific miRNAs by exosomes and their release from melanocytes after exposure to UVR have activities in inducing these cells into premature senescence.

A zebrafish model for nevus regeneration

A zebrafish model for nevus regeneration

IGFBP7 Is Not Required for B-RAF-Induced Melanocyte Senescence

Induction of senescence permanently restricts cellular proliferation after oncogenic stimulation thereby acting as a potent barrier to tumor development. The relevant effector proteins may therefore be fundamental to cancer development. A recent study identified IGFBP7 as a secreted factor mediating melanocyte senescence induced by oncogenic B-RAF, which is found commonly in cutaneous nevi. In contrast to the previous report, we demonstrate that B-RAF signaling does not induce IGFBP7 expression, nor the expression of the IGFBP7 targets, BNIP3L, SMARCB1, or PEA15, in human melanocytes or fibroblasts. We also found no correlation between B-RAF mutational status and IGFBP7 protein expression levels in 22 melanoma cell lines, 90 melanomas, and 46 benign nevi. Furthermore, using a lentiviral silencing strategy we show that B-RAF induces senescence in melanocytes and fibroblasts, irrespective of the presence of IGFBP7. Therefore, we conclude that the secreted protein IGFBP7 is dispensable for B-RAF(V600E)-induced senescence in human melanocytes.

Cell signaling

Oncogene-induced senescence acts as a barrier against tumour formation and has been implicated as the mechanism preventing the transformation of benign melanocytic lesions that frequently harbour oncogenic B-RAF or N-RAS mutations. In the present study we systematically assessed the relative importance of the tumour suppressor proteins p53, p21(Waf1), pRb and p16(INK4a) in mediating oncogene-induced senescence in human melanocytes. We now show that oncogenic N-RAS induced senescence in melanocytes is associated with DNA damage, a potent DNA damage response and the activation of both the p16(INK4a)/pRb and p53/p21(Waf1) tumour suppressor pathways. Surprisingly neither the pharmacological inhibition of the DNA damage response pathway nor silencing of p53 expression had any detectable impact on oncogene-induced senescence in human melanocytes. Our data indicate that the pRb pathway is the dominant effector of senescence in these cells, as its specific inactivation delays the onset of senescence and weakens oncogene-induced proliferative arrest. Furthermore, we show that although both p16(INK4a) and p21(Waf1) are upregulated in response to N-RAS(Q61K), the activities of these CDK inhibitors are clearly distinct and only the loss of p16(INK4a) weakens senescence. We propose that the ability of p16(INK4a) to inhibit the cyclin D-dependent kinases and DNA replication, functions not shared by p21(Waf1), contribute to its role in senescence. Thus, in melanocytes with oncogenic signalling only p16(INK4a) can fully engage the pRb pathway to alter chromatin structure and silence the genes that are required for proliferation.

Oncogene-Induced Senescence Does Not Require the p16INK4a or p14ARF Melanoma Tumor Suppressors

Oncogene-induced senescence is considered to act as a potent barrier to cell transformation, and has been seen in vivo during the early stages of tumor development. Human nevus cells frequently express oncogenic N-RAS or B-RAF, and are thought to be permanently growth arrested. Many studies have suggested that the p16(INK4a) and, to a lesser extent, the p14ARF tumor suppressor proteins act as critical triggers of oncogene-induced senescence in nevi, and thus these proteins represent major inhibitors of progression to melanoma. There have also been reports, however, showing that p16(INK4a) and/or p14ARF is not sufficient to execute the oncogene-induced senescence program. In this study, we examined the impact of melanoma-associated N-RAS(Q61K) on melanocyte senescence and utilized RNA-interference vectors to directly assess the individual contribution of human p14ARF and p16(INK4a) genes to the N-RAS-induced senescence program. We formally show that cultured human melanocytes can initiate an effective oncogene-mediated senescence program in the absence of INK4a/ARF-encoded proteins. Our data are consistent with observations showing that senescent nevus cells do not always express p16(INK4a), and highlight the need to thoroughly explore INK4a/ARF-independent molecular pathways of senescence in human melanocytes.

The relative contributions of the p53 and pRb pathways in oncogene-induced melanocyte senescence

Oncogene-induced senescence acts as a barrier against tumour formation and has been implicated as the mechanism preventing the transformation of benign melanocytic lesions that frequently harbour oncogenic B-RAF or N-RAS mutations. In the present study we systematically assessed the relative importance of the tumour suppressor proteins p53, p21(Waf1), pRb and p16(INK4a) in mediating oncogene-induced senescence in human melanocytes. We now show that oncogenic N-RAS induced senescence in melanocytes is associated with DNA damage, a potent DNA damage response and the activation of both the p16(INK4a)/pRb and p53/p21(Waf1) tumour suppressor pathways. Surprisingly neither the pharmacological inhibition of the DNA damage response pathway nor silencing of p53 expression had any detectable impact on oncogene-induced senescence in human melanocytes. Our data indicate that the pRb pathway is the dominant effector of senescence in these cells, as its specific inactivation delays the onset of senescence and weakens oncogene-induced proliferative arrest. Furthermore, we show that although both p16(INK4a) and p21(Waf1) are upregulated in response to N-RAS(Q61K), the activities of these CDK inhibitors are clearly distinct and only the loss of p16(INK4a) weakens senescence. We propose that the ability of p16(INK4a) to inhibit the cyclin D-dependent kinases and DNA replication, functions not shared by p21(Waf1), contribute to its role in senescence. Thus, in melanocytes with oncogenic signalling only p16(INK4a) can fully engage the pRb pathway to alter chromatin structure and silence the genes that are required for proliferation.

Epigenetic regulation of senescence in human Melanocytes.

Epigenetic regulation of senescence in human Melanocytes.

P16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression.

The melanoma susceptibility locus cyclin-dependent kinase inhibitor 2A encodes two unrelated cell growth inhibitors, p16 and alternative reading frame (ARF). In fibroblasts, both proteins are implicated in cellular senescence, a key barrier to tumor development. The p16 coding sequence is more often mutated in melanoma families than is the ARF sequence. To investigate the role of p16 in melanocytes, we assessed aspects of growth, apoptosis, and immortalization in melanocytes cultured from two melanoma patients, both of whom had two inactive p16 alleles but functional ARF. Growth and senescence were evaluated by cumulative population-doubling curves, and apoptosis by terminal deoxytransferase labeling. Expression of p53 and p21, which are associated with fibroblast senescence, was assessed by immunoblotting. Amphotropic retroviruses were used to transfer exogenous gene sequences into the melanocytes. Both melanocyte cultures showed high rates of apoptosis, which were reduced when the cells were grown in the presence of keratinocyte feeder cells or human stem cell factor plus endothelin 1. With these growth factors, both cultures proliferated for 45-55 net population doublings, markedly longer than the maximum of 10 net population doublings of normal adult human melanocytes in similar media, indicating impaired senescence. One of the cultures developed chromosomal aberrations, with numerous dicentric chromosomes at senescence, consistent with telomere dysfunction. p53 and p21 levels were not elevated in senescent normal melanocytes but were elevated in senescent p16-deficient melanocytes. Interference with p53 function by transfer of human papillomavirus 16-E6 further extended the lifespan of p16-deficient melanocytes. Human telomerase reverse transcriptase was sufficient to immortalize both these cell strains but not normal melanocytes. Normal senescence in human melanocytes requires p16 activity. p53 contributes to a delayed form of senescence that requires telomere shortening, in p16-deficient melanocytes. These findings provide some basis for the role of p16 in melanoma susceptibility.

Human melanocyte senescence and melanoma susceptibility genes.

The molecular mechanisms and biology of cellular senescence in human melanocytes are discussed, including similarities to and differences from senescence in fibroblasts and other cell lineages. Special reference is made to the fact that the known melanoma susceptibility genes in the human, Inhibitor A of [cyclin-dependent] kinase 4-alternative reading frame (INK4A-ARF) and cyclin-dependent kinase 4, are involved in the regulation of cellular senescence, and possible reasons why this should be so. Based on the evidence including growth and survival kinetics of human and mouse melanocytes carrying germline deficiencies in the INK4A sequence, it is suggested that an 'M0' or p16/RB-dependent form of senescence may be particularly important in melanocytes. A speculative model is proposed, relating current concepts of early melanoma progression to the processes of cellular senescence and immortalization. This includes the suggestion that moles or nevi are senescent clones of melanocytes.